Substantial improvement in the efficiency of nucleotide sequencing is needed if the goals of the human genome sequencing project are to be realized. Improvements in sequencing technology would also provide substantial benefit to molecular genetics by liberating creative scientists from the repetitive, but highly informative task of sequencing newly isolated DNAs of interest.
A potentially efficient method of sequencing by current technology is by primer walking. By this technique, priming an enzymatic sequencing reaction within a segment of known sequence (such as vector sequence) is used to extend the sequence into the unknown region. The newly determined sequence in turn is used to select a primer to extend the sequence further, and this process is repeated until the sequence of the entire molecule has been determined. Advantages of primer walking are that the entire sequence can be determined on a single preparation of template DNA without subcloning, and the sequence can be determined in the minimum number of sequencing reactions.
A disadvantage of primer walking has been the inconvenience and expense of having to synthesize a primer for each sequencing reaction. An improvement in priming methods which would eliminate this disadvantage would represent an important advance in the art.